【摘要】：抗腫瘤抗生素在臨床中廣泛使用,但存在藥物毒副作用及腫瘤細(xì)胞多耐藥性等問(wèn)題,為了解決這一難題,尋找結(jié)構(gòu)新穎的抗腫瘤化合物,本研究采用活性追蹤的方法開(kāi)展了抗腫瘤土壤放線菌篩選及對(duì)兩株活性放線菌次級(jí)代謝產(chǎn)物的研究工作。以人肺癌細(xì)胞A549和人結(jié)腸癌細(xì)胞HCT-116為篩選模型,采用CCK-8方法從土壤放線菌中篩選出兩株活性菌,經(jīng)16S r RNA初步鑒定均為鏈霉菌,分別命名為Streptomyces sp.HS-NF-1006和Streptomyces sp.HS-NF-853。結(jié)合抗腫瘤模型篩選結(jié)果和菌株在不同培養(yǎng)基中的生長(zhǎng)狀況,最終選定葡萄糖1%,可溶性淀粉4%,酵母抽提物0.4%,麥芽提取物0.1%,CaCO_3 0.2%,FeSO_4·7H_2O 0.1%,MnCl_2·4H_2O 0.1%為Streptomyces sp.HS-NF-1006的最適發(fā)酵培養(yǎng)基;選定葡萄糖1%,可溶性淀粉4%,酵母抽提物0.4%,麥芽糊精1%,0.1 ml/L(Fe SO_4·7H2O 0.1%,MnCl_2·4H_2O 0.1%,ZnSO_4·7H_2O 0.1%)為Streptomyces sp.HS-NF-853的最適發(fā)酵培養(yǎng)基。利用50 L發(fā)酵罐分別對(duì)兩株活性菌進(jìn)行放大發(fā)酵。所得發(fā)酵液經(jīng)前處理后,利用硅膠、凝膠、HPLC等分離方法對(duì)兩株活性菌的發(fā)酵產(chǎn)物進(jìn)行分離,最終得到9個(gè)化合物,利用核磁共振和質(zhì)譜等分析方法進(jìn)行結(jié)構(gòu)鑒定,從菌株HS-NF-1006提取到3個(gè)化合物1-3,均為蒽環(huán)類(lèi)化合物,其中化合物1為新結(jié)構(gòu)化合物,命名為13-oxy-13-decarbony-6′′-cyano-6′′-deoxy-TAN-1120;從菌株HS-NF-853提取到6個(gè)化合物4-9,其中化合物4是一個(gè)結(jié)構(gòu)新穎度較高的四氫苯并呋喃酮化合物,命名為Strefuranone A。對(duì)所得到化合物進(jìn)行抗腫瘤活性評(píng)價(jià),化合物1對(duì)結(jié)腸癌細(xì)胞HCT-116的IC50為0.0978μM,對(duì)人肺癌細(xì)胞A549的IC_50為0.08μM,化合物2-9未表現(xiàn)抗腫瘤活性。
[Abstract]:Antineoplastic antibiotics are widely used in clinic, but there are some problems, such as side effects of drugs and multi-drug resistance of tumor cells. In order to solve this problem, find novel antitumor compounds. In this study, the screening of actinomycetes in antitumor soil and the secondary metabolites of two strains of actinomycetes were studied by activity tracking method. Using human lung cancer cell line A549 and human colon cancer cell line HCT-116 as screening models, two strains of active bacteria were screened from soil actinomycetes by CCK-8. They were identified as Streptomyces by 16s r RNA. Named Streptomyces sp.HS-NF-1006 and Streptomyces sp.HS-NF-853., respectively. Combined with the screening results of antitumor model and the growth of the strain in different media, glucose 1%, soluble starch 4%, yeast extract 0.4%, malt extract 0.1%, CaCO3 0.2% were selected. FeSO_ 4.7H _ 2O 0.1%, MnCl2 路4H _ 2O 0.1% is the optimum fermentation medium for Streptomyces sp.HS-NF-1006. Glucose 1%, soluble starch 4%, yeast extract 0.4%, maltodextrin 1%, 0.1 ml/L (Fe SO_ 4 路7H2O 0.1%, MnCl2 路4H2O 0.1%), ZnSO_ 4.7H _ 2O 0.1%) was the most suitable fermentation medium for Streptomyces sp.HS-NF-853. Two strains of active bacteria were amplified and fermented in 50 L fermenter. After pretreatment, the fermentation products of the two active bacteria were separated by silica gel, gel and HPLC, and nine compounds were obtained. The structure of the fermentation broth was identified by nuclear magnetic resonance (NMR) and mass spectrometry (MS). Three compounds 1 / 3 were extracted from strain HS-NF-1006, all of which were anthracycline compounds, in which compound 1 was a new structural compound named as 13 脳 13 / decarbony / 6 / cyano / 6 / deoxyto1120; Six compounds 4 鈮，

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